This invention relates generally to the field of antimicrobial therapy. In particular, the present invention relates to methods and products useful in inhibiting the growth of bacteria or other microbes. In addition, this invention relates to identifying loci in bacteria or other microbes which affect antibiotic or antimicrobial susceptibility and to the production of bacterial strains useful in the field of antimicrobial therapy.
Antibiotic or antimicrobial substances have long been used to inhibit the growth of bacteria or other microbes and to treat bacterial or microbial infections in humans, other animals, and in tissue culture. The use of antibiotics or antimicrobials in a treatment regimen, however, has the undesirable effect of selecting for bacteria or other microbes which are resistant to those antibiotics or antimicrobials which are administered or applied. As a result, treatment regimens can be adversely affected or, in some cases, rendered ineffective. This necessitates a continual search for new antibiotics and antimicrobials.
Of particular interest is the discovery of bacteria which express a multiple antibiotic resistance phenotype (Mar). This phenotype entails simultaneous resistance to a multiplicity of antibiotics which are unrelated in chemical structure. The appearance of such bacteria and infections by such bacteria greatly increase the difficulty of identifying effective antibiotics and treating infections in humans or other animals.
Multiple antibiotic resistance in bacteria is most commonly associated with the presence of plasmids which contain one or more resistance genes, each encoding a single antibiotic resistance phenotype (Clewell 1981; Foster 1983). Multiple antibiotic resistance associated with the chromosome, however, has been reported in Klebsiella, Enterobacter, Serratia (Gutmann et al. 1985), Neisseria (Johnson and Morse 1988), and Escherichia (George and Levy 1983a).
Bacteria expressing the multiple antibiotic resistance phenotype can be isolated by selecting bacteria with a single antibiotic and then screening for cross-resistance to structurally unrelated antibiotics. For example, George and Levy initially described a chromosomal multiple antibiotic resistance system which exists in Escherichia coli and which can be selected by a single drug, e.g., tetracycline or chloramphenicol (George and Levy 1983a). In addition to resistance to the selective agents, the Mar phenotype includes resistance to structurally unrelated agents, including nalidixic acid, rifampin, penicillins, and cephalosporins (George and Levy 1983); more recently, resistance to the fluoroquinolones has been described (Cohen et al. 1989).
The expression of a Mar phenotype, conferring substantially increased, simultaneous and coordinated resistance to a multiplicity of structurally unrelated compounds, appears to involve coordinated changes in the expression of a multiplicity of loci. This has been demonstrated in Mar phenotype bacteria of the species E. coli (Cohen et al. 1989). Such coordinated control of the expression of a multiplicity of loci implies the existence of an operon which directly or indirectly regulates the expression of the multiplicity of loci directly responsible for the Mar phenotype. One locus in one such operon was identified in E. coli and named marA by George and Levy (George and Levy 1983b).
Prior to the present invention, however, no multiple antibiotic resistance (mar) operon had been isolated or cloned. In addition, no mar operon had been characterized as to its structure and operation so as to enable the use of such an operon or its fragments for diagnostic, therapeutic or experimental purposes. Finally, the several other contributions to the field of antibacteriology in the claims were unavailable to those skilled in the art prior to the present invention.
The present invention relates generally to developing and evaluating antibiotic treatments effective against bacteria possessing a multiple antibiotic resistance (mar) operon. Because the expression of such an operon causes bacteria to become simultaneously resistant to a multiplicity of structurally unrelated antibiotics, it is a general object of the present invention to provide methods and compositions useful in combating bacteria possessing a mar operon or exhibiting a Mar phenotype. It is one particular object of the present invention to provide tests for compositions which are effective against bacteria expressing a Mar phenotype but which do not induce the expression of a mar operon, or which inhibit the expression of a mar operon. To this end, it is also an object of the present invention to provide cloned nucleotide sequences, as well as bacterial cells expressing such sequences, which are useful in performing such tests and in investigating bacterial multiple antibiotic resistance operons.
The present invention provides cloned bacterial mar operons and cloned fragments thereof. In particular, a cloned repressor locus and a cloned activator locus of a mar operon, as well as cloned loci encoding anti-sense transcripts to the repressor and activator loci, are provided. Using such clones, substantially pure repressor protein and substantially pure activator protein are provided. In addition, using such clones, isolated nucleotide sequences, either sense or anti-sense to those loci, are provided. These sequences are useful as probes for substantially homologous loci in other species including bacteria, fungi, parasites, and animal cells and are useful for altering the expression of a Mar phenotype in bacteria, either by encoding repressor or activator proteins or by encoding anti-sense transcripts which inhibit the expression of either a mar repressor or mar activator locus.
The present invention also provides cloned nucleotide sequences in which the regulatory region of a mar operon has been operably joined to a marker locus. Such sequences are useful in assaying the effect of compositions on the transcription of a mar operon.
The present invention also provides methods for evaluating the antibiotic effectiveness of compositions by assaying their effects upon the transcription of a mar operon or upon the activity of proteins encoded by a mar operon. In particular, the present invention provides methods for assessing the ability or inability of a composition to inhibit the activity of a mar repressor, to enhance the activity of a mar repressor, or to inhibit the activity of a mar activator. Compositions which enhance the activity of a mar repressor or inhibit the activity of a mar activator will be useful either alone or in combination with antibiotics to combat bacteria. A method of treatment for bacterial infections using a combination of such compositions along with antibiotics is thus provided.
The present invention also provides methods for evaluating the antibiotic effectiveness of compositions by assaying their effects on bacteria in which the expression of a mar operon has been substantially increased and on bacteria in which the expression of a mar operon has been substantially decreased. To this end, methods of producing such bacteria and such bacteria themselves are provided.
The present invention also provides tests for identifying loci in bacteria which are subject to regulation, directly or indirectly, by a mar operon. Because such loci may be involved in the expression of a Mar phenotype, their identification will be useful in developing antibiotic compositions which affect the products or expression of those loci.
The present invention also provides cloned bacterial loci and fragments thereof which are subject to mar operon regulation and which, therefore, form part of a mar regulon. Using such clones, substantially pure protein encoded by these loci are provided. In addition, using such clones, isolated nucleotide sequences, either sense or anti-sense to these loci, are provided. These sequences are useful as probes for substantially homologous loci in other species including bacteria, fungi, parasites, and animal cells and for altering the expression of a Mar phenotype in bacteria.